The long-term objective of this research plan is to use modern methods in molecular and structural biology to gain a detailed understanding of the intermediate conformation of the HIV-1 envelope glycoprotein complex (gp120/gp41) and to relate this understanding to the biology of viral entry and to the rational design of potential vaccine immunogens. The focus of this project is on defining the essential structural and physicochemical features of the gp41 ectodomain that is thought to control the key process of HIV-1 membrane fusion and may serve as a target for humoral immunity. The Specific aims of this research are: (1) To use accumulated knowledge of the gp41 ectodomain structure to trap its conformation in a putative intermediate state by destabilization of a fusion-active helical-hairpin structure, and to determine the biophysical and structural properties of the intermediate gp41 model, using techniques including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, in order to understand the factors that influence the folding, thermodynamics, and conformation of gp41. (2) To introduce structural determinants for the intermediate gp41 fold into the gp120/gp41 complex, and to characterize the biological properties of engineered envelope glycoproteins, using assays including protein biosynthesis, soluble CD4-induced shedding, viral infectivity, and neutralization sensitivity, in order to understand the role of these structural determinants in a functional term. (3) To extend studies of 'fusion-competent' HIV vaccine immunogens to the engineered envelope glycoproteins, in order to understand the basis for the broad neutralization of primary HIV-1 isolates by the "fusion-competent" immunogens. This research represents a multidisciplinary approach to elucidating the role of the gp41 conformation in eliciting a broadly neutralizing antibody response. As a result, this work will provide new insights into the mechanism of HIV-1 entry and guide efforts to target this process for vaccine and antiviral drug development.